Immunoassays have become a useful diagnostic tool for detecting the presence or amount of an analyte in a test sample. Various forms of immunoassays, as well as the reagents and procedures necessary to perform such assays, are well known in the art.
One form of a conventional solid-phase immunoassay is a "sandwich assay" which involves contacting a test sample suspected of containing an analyte with a substantially solid inert plastic, latex or glass bead or microparticle, or other support material which has been coated with a protein or another substance capable of binding the analyte to the surface of the support. The analyte and the protein or substance capable of binding the analyte are commonly referred to as a "binding pair" or individually known as "binding members", and a support material coated with a binding member is variably referred to as a "solid phase reagent". After the analyte is bound to the support material the remaining test sample is removed from the support and the analyte bound support material is treated with a second binding member. The second binding member can be conjugated to a signal generating group such as an enzyme, a fluorophore or a chemiluminescent label and collectively, the binding member/signal generating group complex is variably referred to as a "conjugate"or "indicator reagent". The conjugate becomes bound to the analyte which is bound on the support and the solid support, having the first binding member, the analyte and conjugate bound thereon is separated from any unbound conjugate, typically with one or more wash steps. In the case of an enzyme immunoassay, an indicator substance, for example, a chromogenic substrate, is added which reacts with the enzyme to produce a color change. The color change can be observed visually, or more preferably by an instrument, to indicate the presence or amount of an analyte in the test sample. For solid phase fluorescence or chemiluminescence immunoassays, fluorescent labeled binding members can be monitored using excitation at an appropriate wavelength, while chemiluminescent labeled binding members can be monitored after a reaction which chemically activates the chemiluminescent label and generates light which can be detected by photometric means.
Immunoassay reagents such as a solid phase reagent or a conjugate are typically manufactured in bulk and small amounts of the bulk reagents are used to perform individual assays. Remaining bulk reagents are then stored for subsequent assays. The stability of these reagents is paramount to providing analytical methods which exhibit precision and uniformity among individual assays. Instability of such reagents provides for unreproducible assay results as well as an increase in the costs of medical services because unstable bulk reagents must be discarded.
Various methods have been used to increase the stability of immunoassay reagents by preserving the integrity and/or activity of the compounds that comprise the reagents. Some methods of preserving immunoassay reagents involve placing additives such as proteins or carbohydrates into solutions that contain the reagents. Another method of preserving assay reagents includes adding reducing agents (variously referred to as "anti-oxidants") to lyophilized assay reagents. Unfortunately, over time, reducing agents are themselves oxidized and consequently provide only short term reagent protection. Chemical crosslinking has also become accepted as a method for stabilizing macromolecules and thereby preserving their integrity and activity.
Chemical crosslinking can effectively be accomplished by intermolecular crosslinking or intramolecular crosslinking wherein molecules having a greater degree of crosslinking are generally more stable than molecules having a lesser degree of crosslinking. Intramolecular crosslinking refers to covalent bonds or crosslinks that are formed within a single multimeric or monomeric chemical entity. Hence, disulphide bonds occurring within an antibody are exemplary of intramolecular crosslinking. On the other hand, intermolecular crosslinking refers to covalent bonds or crosslinks that are formed between more than one distinct chemical entity such as the bonds which are formed when one compound is conjugated to another. Accordingly, an immunoassay's indicator reagent comprising, for example, an antibody attached or conjugated to an enzyme, is exemplary of intermolecular crosslinking. Additionally, an immunoassay's solid phase reagent or an affinity chromatography gel comprising an antibody bound to a chromatographic gel are further examples of intermolecular crosslinking. While intermolecular crosslinking, as exemplified above, is an effective means of conjugating one chemical entity to another, generally, the degree of crosslinking is minimal and the stability of compounds conjugated in this manner is seldom enhanced.
Crosslinking a chemical entity through multipoint intermolecular crosslinking, however, can greatly enhance the compound's stability. Multipoint intermolecular crosslinking typically results in the formation of a plurality of bonds between a crosslinking agent and the compound which is crosslinked. Such crosslinking is most commonly associated with the bonds formed between a soluble entity such as, for example, a polymer and a protein such as, for example, an enzyme.
Examples of intramolecular and intermolecular crosslinking have previously been described. For example, Wong et al., Enzyme Microb. Technol., vol 14, pg 866-874 (1992); generally outlines techniques and reagents for intramolecularly and intermolecularly crosslinking compounds. Additionally, U.S. Pat. No. 4,652,524 and U.S. Pat. No. 4,657,853 disclose the crosslinking of multiple enzymes to a polymer, and further crosslinking the polymeric enzyme to a binding member. European Patent Application No. 0 049 475 discloses a method for multipoint intermolecular crosslinking an enzyme with a soluble polymer. Unfortunately, however, the aforementioned methods require harsh conditions to effect crosslinking, lack control over the crosslinking process, and/or result in randomly polymerized protein aggregates which are often non-soluble. Moreover the biological performance of the crosslinked entity is often negatively affected as manifested by, for example, lower binding affinities, diminished enzymatic turnover, recognition impairment by specific ligands, and the like.